Research will continue on the long-term goal of elucidating the molecular architecture of a-crystallin, the major protein component of the mammalian lens, and defining the mechanism of its chaperone function. The specific goals for the next funding period are to undertake a detailed thermodynamic analysis of the interactions between c_-crystallins and 13- crystallins, to investigate post-translational modifications that modulate these interactions, and to define the global structural and dynamic features of the native _-crystallin oligomer. The proposed research will definitively test the paradigm that a-crystallins maintain lens transparency through the recognition and binding of unfolding lens proteins. The results will bridge the knowledge gap between the rapidly developing understanding of the temporal trajectories of individual protein components and the consequences on their molecular interactions. The achievements of the previous funding period, including the determination of the folding pattern of the a-crystallin domain and the development of a mechanistic model of aA-crystallin chaperone function provide the bases for the proposed studies. Four specific aims are designed to address two fundamental questions: i) What is the energy threshold required to trigger the binding of 13-crystallin to ct-crystallin and is this threshold crossed in age-related modifications? 2) How is this interaction modulated by the co-oligomerization of a-crystallin subunits and by phosphorylation of aB-crystallin? For this purpose, site-directed mutants of selected 13-crystallins will be constructed and characterized with respect to their stability and binding affinity to a-crystallin. Structural analysis will be performed using newly developed Electron Paramagnetic Resonance distance measurement methods on the 50A scale. Phosphorylation-mimic substitutions will be introduced in ctB-crystallin and their effects on the structure, dynamics and binding to 13-crystallins investigated. A unique combination of molecular biology and biophysical methods will be used to address a problem of central importance to the understanding of the molecular basis of lens transparency. To the extent that modifications of 13- crystallins play a role in age-related opacity, the proposed studies are of fundamental biomedical importance. More generally, a growing number of pathologies have been traced to protein aggregation and failures of the chaperone machinery. The anticipated results will elucidate common molecular events associated with these diseases.